Enhancement of gefitinib-induced growth inhibition by Marsdenia tenacissima extract in non-small cell lung cancer cells expressing wild or mutant EGFR

Abstract

Background: Non-small cell lung cancer (NSCLC) expressed high levels of epidermal growth factor receptor (EGFR). Gefitinib (Iressa) has demonstrated clinical efficacy in NSCLC patients harboring EGFR mutations or refractory to chemotherapy. However, most of NSCLC patients are with wild type EGFR, and showed limited response to gefitinib. Therefore, to develop new effective therapeutic interventions for NSCLC is still required. Our previous study showed Marsdenia tenacissima extract (MTE) restored gefitinib efficacy in the resistant NSCLC cells, but whether MTE acts in the gefitinib-sensitive NSCLC cells is the same as it in the resistant one is unknown.

Methods: Dose response curves for gefitinib and MTE were generated for two sensitive NSCLC cell lines with mutant or wild type EGFR status. Three different sequential combinations of MTE and gefitinib on cell growth were evaluated using IC50 and Combination Index approaches. The flow cytometric method was used to detect cell apoptosis and cell cycle profile. The impact of MTE combined with gefitinib on cell molecular network response was studied by Western blotting.

Results: Unlike in the resistant NSCLC cells, our results revealed that low cytotoxic dose of MTE (8 mg/ml) combined gefitinib with three different schedules synergistically or additively enhanced the growth inhibition of gefitinib. Among which, MTE→MTE+gefitinib treatment was the most effective one. MTE markedly prompted cell cycle arrest and apoptosis caused by gefitinib both in EGFR mutant (HCC827) and wild type of NSCLC cells (H292). The Western blotting results showed that MTE→MTE+gefitinib treatment further enhanced the suppression of gefitinib on cell growth and apoptosis pathway such as ERK1/2 and PI3K/Akt/mTOR. This combination also blocked the activation of EGFR and c-Met which have cross-talk with each other. Unlike in gefitinib-resistant NSCLC cells, MTE alone also demonstrated certain unexpected modulation on EGFR related cell signal pathways in the sensitive cells.

Conclusion: Our results suggest that MTE is a promising herbal medicine to improve gefitinib efficacy in NSCLC regardless of EGFR status. However, why MTE acted differently between gefitinib-sensitive and -resistant NSCLC cells needs a further research.

Figure 1

Treatment of non-small cell lung cancer cells with gefitinib or MTE (M. tenacissima extract) reduced their proliferation potential. (A) H292 and HCC827 cells were treated with the indicated concentrations of gefitinib for 72 h. (B) H292 and HCC827 cells were treated with the indicated concentrations of MTE for 72 h. Cells viability was determined by using the MTT assay as described in the Methods section. The data are expressed in terms of percent of control cells as the means ± SEM. The experiments were repeated at least three times. ^*P < 0.05, ^**P < 0.01 vs. control group.

Figure 2

MTE (M. tenacissima extract) enhances gefitinib induced cytotoxicity in gefitinib-sensitive non-small cell lung cancer cell lines. H292 (A) or HCC827 (B) cells were incubated with increasing concentrations of gefitinib and MTE alone or different schedule combinations. Treatment schedule: (1) M → M + G, MTE pretreated for 12 h, then M + G (MTE + gefitinib) for another 72 h. (2) M + G, MTE and gefitinib concomitantly treated for 72 h. (3) G → G + M, gefitinib pretreated for 12 h, then G + M (gefitinib + MTE) for another 72 h. Growth assays were performed by MTT assay and IC50 values were calculated by Graphpad Prism 5.0 software (San Diego, CA, USA). CI values for the combinations of gefitinib and MTE in H292 (C) and HCC827 (D) cells were calculated using the Calcusyn software (Cambridge, UK), as described in the Methods section. Each data point represented the mean of 3 to 4 replicates; r values for all curves were >0.95. ^*P < 0.05, ^**P < 0.01 vs. control group.

Figure 3

MTE (M. tenacissima extract) enhances gefitinib induced delay in cell cycle in non-small cell lung cancer cell lines H292 (A) and HCC827 (B). Cells were treated with 1 μM gefitinib, 8 mg/ml MTE, and their combination (MTE → MTE + Gef, M → M + G) in 1% FBS culture medium for 72 h, then harvested, ethanol fixed and labeled with PI for the analysis of cell cycle by FACS analysis. Each data presented as the means ± SEM of three experiments. ^*P < 0.05; ^**P < 0.01vs vehicle control group, ^aP < 0.05 vs MTE treated group, ^bP < 0.05 vs gefitinib treated group.

Figure 4

MTE (M. tenacissima extract) prompts apoptosis induced by gefitinib in non-small cell lung cancer cells. (A) H292 and HCC827 cells were treated with 1 μM gefitinib, 8 mg/ml MTE, and their combination (MTE → MTE + Gef, M → M + G) in 1% FBS culture medium for 72 h, then harvested and labeled with Annexin V-PI for the analysis of apoptotic cells by FACS analysis. The lower right quadrant and the upper right quadrant of the FACS histograms indicate the percentage of early and late apoptotic cells, respectively. (B) Total percentages of apoptotic cells in each treatment group are summarized with data presented as the means ± SEM of three experiments. ^*P < 0.05; ^**P < 0.01vs vehicle control group, ^aP < 0.05 vs MTE treated group, ^bP < 0.05 vs gefitinib treated group.

Figure 5

MTE (M. tenacissima extract) enhances the inhibition of gefitinib on PI3K/Akt/mTOR and ERK1/2 signaling cascade in non-small cell lung cancer cells. H292 and HCC827 cells were treated with 1 μM gefitinib, 8 mg/ml MTE, and their combination (MTE → MTE + Gef, M → M + G) in 1% FBS culture medium for 6 h. Cells were stimulates with 10 ng/ml EGF for 15 min before harvest. Cells were lysed and cellular extracts (20 μg protein) were separated on SDS-PAGE gel and transferred to PVDF membranes. (A) Membranes were probed with PI3K, phospho-PI3K, Akt and phospho-Akt; (B) Membranes were probed with mTOR, phospho-mTOR, ERK1/2 and phospho-ERK1/2. Blots are representatives of three independent experiments. Relative density of proteins were calculated and normalized based on β-tubulin or β-actin.

Figure 6

MTE (M. tenacissima extract) combined with gefitinib reduces EGFR and Met crosstalk in non-small cell lung cancer cells. H292 and HCC827 cells were treated with 1 μM gefitinib, 8 mg/ml MTE, and their combination (MTE → MTE + Gef) in 1% FBS culture medium for 6 h. Cells were stimulates with 10 ng/ml EGF for 15 min before harvest. Cells were lysed and cellular extracts (20 μg protein) were separated on SDS-PAGE gel and transferred to PVDF membranes. Membranes were sequentially probed with Met, phospho-Met, EGFR and phospho-EGFR. β-tubulin was served as a loading control. Blots are representatives of three independent experiments.